Titin, the largest protein known, functions as a complex molecular spring that is a dominant contributor to passive myocardial stiffness. Importantly, titin?s stiffness can be tuned post-transcriptionally (by varying the expression ratio of the stiff N2B and more compliant N2BA titin isoforms) and post-translationally (e.g., via changes in protein kinase G (PKG) phosphorylation of titin). Titin?s stiffness is increased in heart failure with preserved ejection fraction (HFpEF), due to deranged titin phosphorylation, in particular hypo-phosphorylation of PKG sites. Currently no effective therapies for HFpEF exist. This application studies potential titin-based treatment options. For this work we have available several animal models of HFpEF: a genetic mouse model in which titin?s spring region is extended to a higher degree and passive stiffness is increased accordingly and pressure-overload (TAC/DOCA) induced mouse and guinea pig models that have diastolic dysfunction and deranged titin phosphorylation. The potential of existing drugs to ameliorate titin-based diastolic stiffening in HFpEF will be addressed in Aims 1 and 2. Metformin is an insulin sensitizing drug that has been shown to improve diastolic function in animal and human studies. Our pilot studies show that metformin rescues diastolic dysfunction and normalizes titin-based stiffness. The phosphodiesterase PDE9A was recently shown to reduce PKG activity and it is known that hypo-phosphorylation of titin?s PKG sites occurs in HFpEF. Hence we also study whether PDE9A inhibition (PDE9Ai) ameliorates diastolic dysfunction. Recent studies showed that titin mutations are causative for dilated cardiomyopathy (DCM), a prevalent form of HF characterized by progressive left ventricular (LV) dilation and systolic dysfunction. Aim 3 seeks to boost understanding of mechanisms by which titin can cause DCM. Through gene targeting we generated the first titin- based mouse model that under baseline conditions develops DCM, the N2BA-PEVK KO. In this model, PEVK sequences that are specific to the N2BA titin isoform were deleted and pilot studies show that this causes severe dilation and a reduction in ejection fraction. DCM-causing mechanisms will be studied at a preclinical stage (2 mo) and after the heart dilates (6 mo), using both an unbiased approach and a candidate approach that focusses on mechanisms that are unique to the N2BA isoform. A rescue experiment is included in which the stress on titin?s spring will be reduced by targeting the titin splicing factor RBM20. With its basic and clinically important research and its in-depth and integrative approach, this proposal seeks to continue our track record of cutting edge titin research. We anticipate that these studies will greatly improve understanding of the roles of titin in HFpEF and provide novel insights in therapeutic options. We also will study a novel mouse model with titin-based DCM and investigate mechanisms by which titin can cause DCM, an area that also needs urgent study. We have supportive pilot data and have an excellent research team in place.